Hepatitis B virus (HBV) is a compact, enveloped DNA virus belonging to the Hepadnavirus family. This virus is the major cause of chronic liver disease and hepatocellular carcinoma world-wide (Hoofnagle (1990) N. Eng. J. Med. 323:337–339). HBV is associated with acute and chronic hepatitis and hepatocellular carcinoma, and may also be a cofactor in the development of acquired immune deficiency syndrome (Dienstag et al. in Harrison's Principles of Internal Medicine, 13th Ed. (Isselbacher et al., eds.) McGraw-Hill, NY, N.Y. (1993) pp. 1458–1483). At least 400 million people worldwide are currently infected with HBV.
There is no known treatment for acute hepatitis. Antiviral therapy with interferon-a has been used for chronic hepatitis, but has met with only partial success, and there are complications from such therapy. Short term therapy with glucocorticoids may be beneficial in conjunction with interferon therapy, but long term treatment is limited by toxicological problems (Dienstag et al. in Harrison's Principles of Internal Medicine, 13th Ed. (Isselbacher et al., eds.) McGraw-Hill, NY, N.Y. (1993) pp. 1458–1483). Thus, emphasis has been placed on prevention through immunization.
New chemotherapeutic agents have been developed which are capable of modulating cellular and foreign gene expression (see, Zamecnik et al. (1978) Proc. Natl. Acad. Sci. (USA) 75:280–284; Zamecnik et al. (1986) Proc. Natl. Acad. Sci. (USA) 83:4143–4146; Goodchild et al. (1988) Proc. Natl. Acad. Sci. (USA) 85:5507–5511). These agents, called antisense oligonucleotides, bind to target single-stranded nucleic acid molecules according to the Watson-Crick rule or to double-stranded nucleic acids by the Hoogsteen rule of base pairing, and in so doing, disrupt the function of the target by one of several mechanisms: by preventing the binding of factors required for normal transcription, splicing, or translation; by triggering the enzymatic destruction of mRNA by RNase H, or by destroying the target via reactive groups attached directly to the antisense oligonucleotide.
Improved oligonucleotides have more recently been developed that have greater efficacy in inhibiting such viruses, pathogens and selective gene expression. Some of these oligonucleotides having modifications in their internucleotide linkages have been shown to be more effective than their unmodified counterparts. For example, Agrawal et al. (Proc. Natl. Acad. Sci. (USA) (1988) 85:7079–7083) teaches that oligonucleotide phosphorothioates and certain oligonucleotide phosphoramidates are more effective at inhibiting HIV-1 than conventional phosphodiester-linked oligodeoxynucleotides. Agrawal et al. (Proc. Natl. Acad. Sci. (USA) (1989) 86:7790–7794) discloses the advantage of oligonucleotide phosphorothioates in inhibiting HIV-1 in early and chronically infected cells.
In addition, chimeric oligonucleotides having more than one type of internucleotide linkage within the oligonucleotide have been developed. Pederson et al. (U.S. Pat. Nos. 5,149,797 and 5,220,007) discloses chimeric oligonucleotides having an oligonucleotide phosphodiester or oligonucleotide phosphorothioate core sequence flanked by nucleotide methylphosphonates or phosphoramidates. Furdon et al. (Nucleic Acids Res. (1989) 17:9193–9204) discloses chimeric oligonucleotides having regions of oligonucleotide phosphodiesters in addition to either oligonucleotide phosphorothioate or methylphosphonate regions. Quartin et al. (Nucleic Acids Res. (1989) 17:7523–7562) discloses chimeric oligonucleotides having regions of oligonucleotide phosphodiesters and oligonucleotide methylphosphonates. Inoue et al. (FEBS Lett. (1987) 215:237–250) discloses hybrid oligonucleotides having regions of deoxyribonucleotides and 2′-O-methyl-ribonucleotides.
Antisense oligonucleotides have been designed which inhibit the expression and/or replication of HBV. For example, antisense oligonucleotides directed against the cap site of HBV mRNA transcribed from the SPII promoter (Goodarzi et al. (1990) J. Gen. Virol. 71:3021–3025; Yao et al. (1994) Nat. Med. J. China 74:125), against the translational initiation site of the S gene (Yao et al. (1994) Nat. Med. J. China 74:125; Reinis et al. (1993) Folia Biologica (Praha) 39:262–269; Goodarzi et al. (1990) J. Gen. Virol. 71:3021–3025); against a portion of the core-pol mRNA encoding the terminal protein region of the viral polymerase (WO 94/24864; Blum et al. (1991) Lancet 337:1230), and against the HBV polyadenylation signal (Wu et al. (1992) J. Biol. Chem. 267:12436–12439) have been designed. In addition, phosphorothioate oligodeoxynucleotides prepared against the 5′ region of the pre-S gene have been shown to inhibit duck HBV replication and gene expression invivo (Offensperger et al. (1993) EMBO J. 12:1257–1262).
A need still remains for the development of oligonucleotides that are capable of inhibiting the replication and expression of HBV whose administration are accompanied by a good prognosis and low or no cellular toxicity.